The long-term objective of this proposal is to generate novel immunogens that are capable of eliciting potent HIV-1 neutralizing antibodies. The HIV-1 infection is initiated by the binding of viral envelope glycoproteins (Envs) to the CD4 receptor. Due to the high degree of conformational flexibility, the native HIV-1 Envs sample many different conformations, from which only some specific conformations are capable of mediating virus infection (designated functional conformations, or functional Envs). To date all Env-based vaccine candidates have generated high titers of anti-Env antibodies, but they failed to induce potent neutralizing antibodies. One possible reason for this failure is that the previous Env-based vaccine candidates all contain a mixture of different conformations of Env, of which only a small population contains functional conformations. Therefore much of the antibody response is likely directed to nonfunctional conformations, which would explain the high titers of the non-neutralizing antibody responses. We hypothesize that functional conformations of Env are the targets for antibodies to neutralize virus infection, and are the better immunogen candidates for inducing potent neutralizing antibody responses. Previous studies showed that monoclonal antibodies (Mabs) directed against CD4-binding site (CD4BS) on Env block HIV-1 binding to the CD4 receptor, but most of anti-CD4BS Mabs do not neutralize primary HIV-1 isolates. This phenomenon indicates that most anti-CD4BS Mabs bind to the Envs that are irrelevant to virus infection (designated nonfunctional Envs). As the Envs recognized by non-neutralizing Mabs are also recognized by the CD4 molecule, the CD4 molecule must recognize both functional and nonfunctional Envs. Therefore the functional Envs would be those that are recognized by CD4, but not by non-neutralizing anti-CD4BS Mabs. For the same reason, the Envs recognized by non-neutralizing Mabs are also recognized by a potent neutralizing anti-CD4BS Mab, IgG1b12. Therefore the Envs that contain neutralizing epitopes at CD4BS would be those that are recognized by IgG1b12, but not by non-neutralizing anti-CD4BS Mabs. In this application, we propose to isolate the functional Env trimers, as well as the Env trimers that contain neutralizing epitopes at CD4BS. These trimers present the functional conformations or the neutralizing epitopes at the CD4BS, therefore would be better immunogens to induce potent neutralizing Ab response against HIV-1 than the native Env trimers that contain both functional and nonfunctional conformations. In addition, since the functional conformations at CD4BS must be conserved to allow interaction with the invariant receptor, Abs elicited against these conformations would be able to neutralize a wide array of primary HIV-1 strains. In R21 part of this proposal, we will 1) examine the percentage of functional conformations existing in four types of soluble Env trimers from three primary HIV-1 isolates. The Env trimer type that contains the highest percentage of functional conformations is expected to best resemble the functional Envs, and will be picked for the further investigation. 2) Purify the functional Env trimers and examine if the functional trimers are recognized better by neutralizing Mabs and less well by non-neutralizing Mabs, and if they are more capable of absorbing neutralizing Abs from sera of HIV-1-infected individuals (PS). In R33 part, we will test the functional Env trimers in animals to determine if they will elicit antibodies that exhibit potent neutralizing activities. The studies in this proposal are expected to provide a proof-of-concept evaluation of our hypothesis that HIV-1 Env in the functional conformations will induce improved antibody responses to neutralize HIV-1. PUBLIC HEALTH RELEVANCE: The purpose of this project is to develop a safe and effective preventive vaccine against HIV/AIDS. The applicant will isolate a functional form of HIV envelope glycoprotein as vaccine candidate. This functional form of HIV envelope glycoprotein is expected to be better than previous HIV vaccine candidates in inducing immune responses against HIV.